This is an instrumentation development proposal for improved electron paramagnetic resonance (EPR) spectroscopy for biomedical research. Specifically, the applicants propose to develop saturation recovery (SR) EPR and SR electron-electron double resonance (ELDOR) apparatus at high microwave frequencies that is tailored for experiments using nitroxide radical spin labels and spin probes, in the context of site-directed spin labeling (SDSL). These studies help to determine the dynamics of molecular structure. SR is often used to study bimolecular collisions of oxygen with spin labels. Measurement of oxygen accessibility in SDSL permits identification of structural motifs of proteins. In this proposal, focus not only is on oximetry but also on the use of SR EPR and SR ELDOR to study slow rotational diffusion of proteins. This laboratory pioneered extension of SR instrumentation to high microwave frequencies. The first SR instrument at Q-band (35 GHz) was developed in the previous funding period, and the first at W-band (94 GHz) in the current funding period. It was discovered that the loop-gap resonator (LGR) yields outstanding performance because of high bandwidth and high efficiency in concentrating the microwave field intensity. In Aim 1 of this proposal, extension to D-band (144 GHz) using an LGR is proposed. The features of the relatively immobilized spin-labeled protein no longer overlap at this frequency, which is highly advantageous. The design is based on an innovative combination of microwave frequency translation and low order multiplication. In Aims 2 and 3, SR technology at W-band (94 GHz) will be enhanced. As in Aim 1, these two aims focus on application to proteins undergoing slow rotational diffusion. In Aim 2, the level of microwave power will increase, the pulses will become shorter, and the dead-time will be decreased: all of which will improve SR EPR and SR ELDOR in the study of slow rotational diffusion. The technique of inversion recovery (IR) will be developed, which in comparison with SR will improve the SNR by two times and improve temporal resolution by about ten times. In Aim 3, another way to excite the spin system is introduced: use of adiabatic rapid passage (ADR) by sweep of the microwave frequency from one point in the spectrum to another to excite the spins. The discovery that LGRs are useful at high microwave frequencies because of high bandwidths is central to Aim 3. A state-of-the-art arbitrary waveform generator (AWG) is an enabling technology for frequency swept ADR excitation. Aims 2 and 3 are expected to be complementary, with the methods of Aim 2 being on a shorter timescale and the methods of Aim 3 providing more uniform excitation over well-defined regions of the spectrum. The engineering team is very experienced and is considered to be a national resource. This proposal contributes to the nation's infrastructure for biomedical research. Developed instruments become available to all researchers through the National Biomedical EPR Center, a P41 Research Resource directed by the PI. [unreadable] [unreadable] [unreadable]